Sample mounts for microcrystal crystallography

ABSTRACT

Sample mounts ( 10 ) for mounting microcrystals of biological macromolecules for X-ray crystallography are prepared by using patterned thin polyimide films ( 12 ) that have curvature imparted thereto, for example, by being attached to a curved outer surface of a small metal rod ( 16 ). The patterned film ( 12 ) preferably includes a tip end ( 24 ) for holding a crystal. Preferably, a small sample aperture is disposed in the film for reception of the crystal. A second, larger aperture can also be provided that is connected to the sample aperture by a drainage channel, allowing removal of excess liquid and easier manipulation in viscous solutions. The curvature imparted to the film ( 12 ) increases the film&#39;s rigidity and allows a convenient scoop-like action for retrieving crystals. The polyimide contributes minimally to background and absorption, and can be treated to obtain desired hydrophobicity or hydrophilicity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. 120 of U.S.application Ser. No. 11/228,455, filed Sep. 19, 2005, to issue as U.S.Pat. No. 7,263,162 on Aug. 28, 2007, which is a continuation under 35U.S.C. 120 and 365(c) of International Application No.PCT/US2004/006088, filed Mar. 22, 2004, which claims the benefit under35 U.S.C. 119(e) of U.S. Application No. 60/455,853, filed Mar. 20,2003.

GOVERNMENT SPONSORSHIP STATEMENT

This invention was made with Government support from the NationalInstitutes of Health (NIH) under grant number GM065981 and the NationalAeronautics and Space Administration (NASA) under grant numberNAG8-1831. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates in general to sample mounts for mountingand manipulating macromolecular and virus crystals and other samples forX-ray crystallography, and methods of using the same.

2. Description of the Background Art

One of the most common ways of mounting crystals for X-ray datacollection and structure determination is to insert them intothin-walled (typically 10 micrometer) glass or quartz capillaries. Thesethin capillaries are X-ray transparent and produce relatively littlebackground scatter. They can be sealed at both ends, providing a stableenvironment for the crystal. This is particularly important for crystalsof proteins and other biological macromolecules, which contain largeamounts of solvent (mostly water) and must be maintained in a constanthumidity environment to preserve their structure and order. Theenvironment of the crystal can be changed inside the capillary. Forexample, crystals can be controllably dehydrated by injecting a smallamount of a saturated salt solution into the capillary. Crystals canalso be soaked inside the capillary in solutions containing drugmolecules, small molecule ligands, and heavy atom compounds. Capillarymounted crystals can be used for data collection from the melting pointof the crystal solvent to well above room temperature. They areparticularly important for crystals that cannot be flash frozen for datacollection without inducing excessive crystal disorder.

In a known technique used to mount protein and other biomolecularcrystals in capillaries, a capillary of diameter comparable to thecrystal diameter is first selected, in order that the final mountedcrystal lie near the center of the capillary to simplify alignment inthe X-ray beam. Next, the sealed end of the capillary is scored andbroken, and some means for producing suction attached to the other,larger diameter end. The open end of the capillary is inserted into theliquid drop in which the crystal resides and a small amount of thisliquid is pulled into the capillary. The capillary is removed from theliquid and a small amount of air is pulled in to move the liquid awayfrom the end. Next, the capillary is inserted back into the liquid dropand the crystal is carefully sucked in. Excess liquid surrounding thecrystal is removed using paper wicks, and the open end of the capillarysealed with wax, grease, etc. The suction device is removed from thelarge diameter end, the capillary is scribed and broken to the desiredlength, the end is sealed and then the capillary is mounted onto a pinor goniometer head, typically using modeling clay, for X-raymeasurements.

Another area where crystal mounting and manipulation is employed is inX-ray cryocrystallography, which is extensively used for crystals ofproteins, protein-nucleic acid complexes and viruses and other crystalsthat are sensitive to radiation damage by X-rays. The development andapplication of cryocrystallographic techniques has had a dramatic impacton the rate at which structures of biological macromolecules andmacromolecular complexes can be solved. Much larger X-ray doses can beabsorbed before radiation damage becomes significant, so that completedata sets can often be collected using a single crystal.

A variety of methods have been used to manipulate mount crystals forflash cooling and cryocrystallographic data collection. Earlyexperiments attached crystals to the ends of glass fibers or placed themon top of miniature glass spatulas. A loop mounting method using lowX-ray absorption materials for the loop is now by far the most widelyused method for manipulating and mounting crystals. Loop cryomountsconsist of a small (10-20 μm) diameter nylon (or metal) line that istwisted to form a loop and then threaded into a small hollow metal rod.This rod is then inserted into a metal or plastic goniometer-compatiblebase. Crystals are retrieved from the mother liquor in which they aregrown by capturing them in the loop, and then they are transferred usingthe loop between one or more solutions including stabilizing solutions,heavy atom compound solutions, solutions containing small molecules,drugs or ligands, or cryoprotectant solutions. Crystals larger than theloop can rest on its surface, while smaller crystals can be trapped inthe liquid film that spans the loop or else adhered to the side of theloop. Loop-mounted crystals are then flash cooled by immersion in liquidnitrogen or propane or by insertion in a cold gas stream.

Loops provide convenient crystal manipulation. By holding crystals inthe liquid film of the loop, potentially damaging contact with hardsurfaces (such as those of alternative mounting tools) is minimized. Theloop itself is flexible enough to make damage due to incidental contactless severe. Loops help minimize thermal mass and maximize surface areafor heat transfer, increasing cooling rates and thus reducingcryoprotectant concentrations needed to prevent hexagonal ice formationwithin and surrounding the crystal. For these reasons loop-based mountshave been chosen as the standard for high-throughput automatedcryocrystallography at synchrotron X-ray beam lines around the world.

The foregoing known techniques for mounting samples in crystallographyapplications have a number of drawbacks. In particular, the capillarymounting technique requires that both the initially sealed and widediameter open end of the capillary must be cut and then subsequentlysealed. These manipulations increase the chance that the capillary willbe broken and the crystal inside damaged. In addition, the minimumcapillary wall thickness that can be used and still provide adequaterobustness for cutting is limited to about 10 micrometers. This in turnfixes the capillary's contribution to background X-ray scatter, whichcan significantly degrade the overall signal-to-noise ratio whenmeasuring very small crystals.

For very small crystals (<50 micrometers), wicking away excess liquidwithout disturbing the crystal is difficult. Residual liquid between thecrystal and capillary wall may have a volume comparable to the crystalvolume (because of the larger surface to volume ratio of smallcrystals), which will increase background scattering of X-rays. Anyresidual liquid between the crystal and capillary also acts with thecurved capillary wall as a distorting lens that makes accurate crystalalignment in the X-ray beam difficult. While some liquid is required tohold the crystal in place against the capillary wall, if there is excessliquid, the crystal may slip relative to the wall during diffractionmeasurements, which can create errors in data analysis.

To ensure that the crystal ends up near the radial center of thecapillary, the capillary diameter must be matched to the crystal size.Consequently, capillaries of many diameters must be stocked. Even with acorrectly sized capillary, the vertical and horizontal position of thecrystal relative to the axis of the X-ray system is poorly controlled,requiring time-consuming alignment for each crystal. Positioning thecrystal at a particular distance, e.g., from the base to be mounted inthe goniometer head requires careful crystal manipulation and carefulcutting of capillaries, which can be very time consuming. Adjustment ofpressure in the suction device during crystal retrieval also requiresconsiderable skill. Because of their large size, capillaries canobstruct the view of the crystal to be mounted, and are difficult tomanipulate in small drops. Contact with the capillary ends and wallsduring retrieval often damages crystals (especially those withplate-like geometries), increasing their mosaicity and degrading theirX-ray diffraction properties. Although possible, retrieving crystalsfrom capillaries for further treatment or measurement is extremelydifficult. Soaking crystals in ligands, drugs or heavy-atom compoundsafter data collection of a native structure can be performed in thecapillary, but is inconvenient and often displaces the crystal.

In cryocrystallography, the use of loop mounts is also problematic. Inparticular, loops are quite flexible, especially those made using 10 μmdiameter nylon line. As a result, loops can bend under liquid andsurface tension forces during crystal retrieval from solution, and theycan bend under the weight of the crystal and surrounding liquid once acrystal is mounted. Because of their irregular aerodynamic profile theycan bend and flutter under the drag forces of the cryostream, slightlybroadening X-ray diffraction peaks for the lowest mosaicity crystals andreducing the maximum diffraction signal-to-noise achieved when crystalmosaicity and incident beam divergence are matched.

In addition, loops provide poor crystal positioning accuracy andreproducibility relative to the X-ray spindle axis. The loop shape for agiven nominal loop diameter is irregular and irreproducible. The looporientation relative to the metal post through which they are threadedis irregular, in part due to the twist of the nylon at their base neededto improve rigidity. Crystal positioning within the loop isirreproducible, especially for very small crystals. The crystal and theloop itself (which gains rigidity from frozen liquid) can shift duringin situ crystal “annealing” or “tempering” protocols that raise thesample temperature near or above the melting point/glass transition ofthe surrounding solvent, necessitating crystal realignment in the X-raybeam.

Loops can also trap significant liquid around the crystal. This liquidcan be difficult to wick away, especially if the crystal is smaller thanthe loop's inner area. Remaining liquid increases background scattering,reducing diffraction signal-to-noise, and increases thermal mass,thereby decreasing cooling rates. Moreover, surrounding liquid hasdifferent freezing properties and thermal expansion behavior than thecrystal and can exert damaging forces during cooling. Frozen surroundingliquid also can make small (less than 50 μm) crystals difficult to imageand align.

The limitations of loops are becoming increasingly apparent ascrystallographers attempt structural studies using smaller and smallercrystals made possible by continuing improvements in X-ray sources,optics and detectors. Initial crystallization trials—especially thosebased on high-throughput robotic screening—usually yield very smallcrystals. Collecting X-ray data from these crystals can provide valuablefeedback early in the growth optimization process, and sometimesimmediately yields useful structural information. Crystal size mayremain small even after substantial optimization of crystal quality,especially in the case of macromolecular complexes and membraneproteins. Despite reduced signal-to-noise and increased radiationdamage, smaller crystals may even be desirable because they flash coolmore rapidly and thus are easier to cryoprotect; they can yield betterdiffraction data sets than larger crystals unless cryoprotectionconditions for the latter are carefully optimized.

For crystals with sizes below 50 μm, loops become extremely difficult touse. Flexibility constraints limit the minimum nylon diameter, which inturn limits the minimum inner loop diameter. Smaller crystals must oftenbe held in a liquid meniscus of larger volume, reducing diffractionsignal-to-noise and making alignment more difficult. Largeliquid-to-crystal volume ratios also limit reductions in thermal massand cooling times.

In view of the foregoing, a need remains for improved techniques formounting microcrystals for use in X-ray crystallography andcryocrystallography applications.

SUMMARY OF THE INVENTION

The present invention addresses the foregoing need through provision ofmicrocrystal sample mounts in which a thin plastic film is employed tosupport a crystal to be analyzed. The film has a thickness of 50 μm orless and is preferably made from polyimide. A key feature of theinvention is that a curvature is imparted to the film to increase itsstructural rigidity substantially. The increase in rigidity enables theuse of thinner films with thicknesses on the order of 3-15 μm (or eventhinner, provided other mount dimensions are correspondingly reduced),which reduces background scattering of the X-rays.

To provide the requisite curvature, a number of techniques can beemployed, depending upon the type of crystallography application. Incryocrystallography applications, where capillary tubes are notemployed, the polyimide film is preferably mounted either to an externalcurved surface of a rod or pin, the internal curved surface of a hollowrod or sleeve, or between both a sleeve and a rod. In these embodiments,the rod and/or hollow sleeve preferably have beveled top ends tomaximize the viewing angle of the sample position so that one can seecrystals and any crystal aperture when the rod, sleeve and mount areangled relative to the horizontal. Alternatively, the film can beattached to a rod having a conical end such that the plastic mount istilted relative to the rod, so that it is easier to scrape a crystal offa flat surface and the crystal is located on axis, which makes alignmenteven easier.

In crystallography applications that use capillary tubes, the curvaturecan also be imparted by making the film large enough that the capillaryitself will impart the requisite curvature when the film is insertedtherein. In this variation, a suitable tool, such as a pair of forceps,can be employed to impart the curvature initially to the film so that itcan be inserted into the capillary or tube.

Polyimide is a good choice for the film material because of itsexcellent mechanical properties; because it has a low density and iscomposed of low atomic number elements so that it scatters X-rays veryweakly; and because its gold hue provides good optical contrast withmacromolecular crystals. The polyimide films are microfabricated to havea tapered tip end for holding a crystal to be examined. This taperingminimizes the volume of the film in the X-ray beam (whose size isusually matched to or smaller than the crystal size) when the plane ofthe film is oriented parallel to the beam. Preferably, a first sampleaperture is disposed at the tip end for reception of the crystal, thoughthe aperture is not essential. This aperture allows the crystal to beprecisely located, and further minimizes the volume of the film in theX-ray beam and thus the background scatter from the film. For mountsintended for cryocrystallography of macromolecular or virus crystalsgrown in solution, a small channel is preferably disposed in the filmthat connects the sample aperture to a larger aperture which facilitateswicking of any excess fluid from the sample aperture with little risk oftouching the crystal. The large aperture also reduces the total area ofpolyimide and thus reduces the fluid resistance and flow disturbancescaused as the mount is moved through a crystal-containing drop. Thechannel and large aperture are not necessary for “dry” crystals ofinorganic or small-molecule organic materials where excess fluid is notan issue.

The film preferably has a small fixed width (5-100 μm, depending on thesize of the crystals to be examined) surrounding the sample aperturethat reduces scattering from the polyimide film (and any adsorbed fluid)when the plane of the film is oriented parallel to the X-ray beam. Anoverall triangular shape of a top portion of the film that is intendedto extend beyond the rod or sleeve to which it is attached, togetherwith a tapered or beveled shape of the rod or sleeve, provides a goodaerodynamic profile that minimizes sample “flutter” in a gas coolingstream (relevant for lowest mosaicity crystals).

The present invention thus provides an alternative to conventional loopsthat retains all of their advantages (including complete compatibilitywith existing and developing technologies for high-throughputcrystallography), but that resolves most if not all of the problems inmounting smaller crystals. Their potential advantages include completelyreproducible sample “loop” sizes down to 3 micrometers or even smallerif thinner, shorter films with larger curvature are used, accurate andreproducible sample positioning, good sample-mount contrast, easierremoval of excess liquid, minimal thermal mass and more rapid flashcooling; reduced background scattering, and easy design customizationand mass production. In addition, the color, rigidity andreproducibility of the mounts should make it feasible to retrievecrystals using robots, with large savings in labor. In contrast, the useof loop type mounts requires that all crystals be retrieved by hand fromcrystallization drops, because loops are floppy and have little contrastwith drop solutions.

BRIEF DESCRIPTION OF THE DRAWINGS

The features and advantages of the present invention will becomeapparent from the following detailed description of a number ofpreferred embodiments thereof, taken in conjunction with theaccompanying drawings, in which:

FIGS. 1A-1D are illustrations of four microcrystal sample mounts thatare constructed in accordance with the preferred embodiments of thepresent invention;

FIGS. 2A and 2B are exploded and assembled views, respectively, of anassembly using one of the preferred sample mounts to position amicrocrystal in a controlled environment capillary or tube using a basecompatible with standard X-ray equipment; and

FIG. 3 is an illustration of a patterned polyimide film that forms theactual crystal mount in the preferred embodiments of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

With reference to FIGS. 1A-1D, several variations of a microcrystalmount 10 are illustrated that are constructed in accordance with fourpreferred embodiments of the invention and are each designedparticularly for use with a goniometer or other standard sampleholder/positioner used in X-ray crystallography. In each embodiment, themount 10 includes a microfabricated plastic film 12 that is attached toa structure having a curved surface such that curvature is imparted tothe film 12. In the embodiment of FIG. 1A, the film 12 is attached byany suitable means, such as adhesive, tape, thermal bonding or the like,to a small diameter cylindrical plastic or metal post 16. As illustratedin FIG. 2A, the post 16 is sized to be inserted into standard plastic ormetal magnetic goniometer head mount 18, such as those sold by HamptonResearch or being developed by the macromolecular crystallographycommunity for high-throughput automated crystallography, or into keyedvariants of these allowing for higher precision alignment of the film 12and the post 16 relative to the goniometer head (not shown). The film 12has a tapered end that tapers to a tip 24 and forms a scoop shape whenattached to the rod 16.

In the embodiment illustrated in FIG. 1B, a hollow metal or plasticsleeve or rod 14 is also employed in conjunction with the rod 16 suchthat the film 12 is held between these two elements. The sleeve 14rigidly attaches the film 12 to an outside curved surface 26 of the rod16 and forces the film 12 to conform to the rod's curvature whileallowing easy assembly. Preferably, a small amount of glue is used toseal the area between the sleeve 14 and the rod 16 and fix the sleeve 14relative to the rod 16. Alternatively, the sleeve 14 can be implementedusing heat-shrinkable tubing that will grip the film 12 between thesleeve 14 and the rod 16. Still further, as illustrated in FIG. 1C, thehollow sleeve 14 can be used by itself such that the film 12 isattached, e.g. by adhesive, tape, thermal bonding, etc. to the insidecurved surface 28 of the hollow sleeve 14. The film 12 can also becrimped to the inside curved surface of the sleeve 14 by collapsing oneside of the sleeve so that it squeezes the film against the other curvedside.

In both of the embodiments illustrated in FIGS. 1A and 1B, the sleeve 14and the rod 16 preferably have beveled top ends 20 and 22, respectively,to maximize the viewing angle of the sample position, which is locatedat the tip end 24 of the polyimide film 12 as discussed in greaterdetail later in conjunction with FIG. 3. An alternative embodiment whichachieves the same goal is illustrated in FIG. 1D in which the rod 16 hasa conical top end 29 to which the film 12 is attached. As shown, thefilm 12 is mounted at an angle relative to the vertical longitudinalaxis of the rod 16 such that the tip end 24 of the film is positioneddirectly over this axis. As a result, alignment of the film 12 is madeeasier. More importantly, the angling of the film 12 positions its scoopshaped tip in such a way that scraping of crystal off of a flat surfaceis made easier.

The curvature of the polyimide film 12 that is imparted by any of thepreferred embodiments dramatically increases its bending rigidity; acylindrically curved piece of paper is much harder to bend than a flatone. This extra rigidity is crucial in allowing the film 12 to be madevery thin, on the order of 3-15 μm, thereby minimizing backgroundscatter from the polyimide film 12 and allowing film patterning to thesmall lateral dimensions required for mounting the smallest crystals. Asjust noted, this curvature also produces a convenient, gentle scoop-likeaction when retrieving crystals and for transferring crystals betweensolutions that minimizes the chance of crystal damage.

For room temperature crystallography applications where the samplecrystal is to be maintained in a controlled environment, a plastic tubeor glass X-ray capillary 30 is slid over the mount 10 and a tip end 32of the tube 30 is then sealed as illustrated in FIG. 2B. In thisembodiment, both the hollow sleeve 14 and the rod 16 need not be used ifdesired. Instead, the polyimide film 12 can be held by a pair of forcepsthat impose the desired curvature on the film 12. The film 12 is theninserted into an X-ray capillary 30 using the forceps. When the forcepsare released, the curvature of the film 12 relaxes. The film width ischosen to be slightly larger than the capillary inside diameter so thatthe film 12 is held in place near the center of the capillary by itsspring-like action. The end 32 of the capillary 30 can then be sealedusing grease or wax. Additional liquid can be inserted into thecapillary before sealing as desired.

In the event that the sleeve 14 and or rod 16 are employed to supportthe film 12, the X-ray capillary 30 with the narrow end cut is carefullypulled over the crystal and down onto the base 18. The base 18 may belubricated with oil or grease and a rubber or silicone washer/O-ringpulled over the end of the capillary to provide an airtight seal withthe capillary. The cylindrical portion over which the capillary ispulled can be an integral part of the base that attaches to the X-rayapparatus, or can be a separate cylindrical piece with a hole throughwhich the rod is inserted into the base, with any gap between rod andthe inside diameter of the hole sealed with grease. The narrow end isthen sealed using grease, wax, glue, etc. Alternatively, the glasscapillary 30 can be replaced with thin walled plastic tubing. Forexample, Advanced Polymers Incorporated of Salem, NH manufacturesregular and low-temperature heat-shrinkable semi rigid transparenttubing with wall thicknesses of 0.0002 inches or 5 micrometers. In thiscase the tubing is pulled over the crystal and onto the base. Sealingboth ends can be done with grease or oil, and/or in the case oflow-temperature heat-shrinkable tubing using a variable temperaturesoldering iron with a custom curved tip. A small plug of any materialcan be inserted in the open end before sealing to improve the seal. Inboth cases, additional liquid can be inserted into the capillary/plastictube before sealing. Also in both cases, the diameter of the capillaryor plastic tube can be much larger than the width of the sample mount,making it easy to slide the capillary or plastic tube over the samplewithout disturbing it.

In all three of the above cases, but with particular ease in the secondand third methods, which are preferred, crystals can be removed from thecapillary/tube after X-ray data collection and subjected to othermanipulations such as soaking in ligand, drug, or heavy atom solutionsand then easily reinserted for additional data collection.

FIG. 3 shows a typical pattern for the polyimide film 12. It should benoted that the film 12 can also be made of any other suitable plastic orother material, but polyimide, which has low density, low atomic numbersof its constituents, and therefore low X-ray absorption and scattering,which is widely used (in the form of KAPTON tape) for temporary windowson X-ray beamlines, and which is readily patterned usingmicrolithographic techniques of the microelectronics industry, is thepreferred choice. In addition, the good contrast between the yellow-goldpolyimide film and a crystal makes locating and aligning the crystaleasier. The film 12 preferably includes a small sample aperture 50 atthe narrow tip end 24 for holding a sample crystal to be analyzed orexamined. The sample aperture 50 is preferably connected via a channel52 to a much larger aperture 54. This structure allows a paper wickinserted into the large aperture 54 to remove excess fluid from aroundthe crystal via the channel 52 with little risk of touching the crystal.The large aperture 54 also reduces the total area of polyimide and thusreduces the fluid resistance and flow disturbances caused as the mountis moved through a crystal-containing drop. Alternatively, neither ofthe apertures 50 and 54, nor the channel 52 need to be employed as acrystal to be examined can also be attached directly to the tip end 24of the film 12, either by means of a droplet of liquid or a polymer, forexample.

The film 12 has a rim 56 of small width surrounding the sample aperture50 that reduces scattering from the polyimide film 12 (and any adsorbedfluid) when the plane of the film 12 is oriented parallel to the X-raybeam. Preferably, the rim 56 has a fixed width of between 5 and 100 μmthat depends on the size of the crystal to be examined. A smallcross-shaped aperture 58 is centered in the film 12 beneath the sampleaperture 52 and the large aperture 54 that can be used to assistautomated alignment. Other alignment apertures can be added to suit thealignment algorithm to be used. The overall triangular shape of a topportion 60 of the film which extends beyond the post 16, together withthe post's tapered shape provides a good aerodynamic profile thatminimizes sample “flutter” in a gas cooling stream (relevant for lowestmosaicity crystals.) The sample aperture 52 can be customized to anyshape so as to simplify mounting of, e.g., rod-shaped crystals. The filmthickness can also be varied, e.g., to produce ridges surrounding thesample aperture 52, to provide a stiffer base, or to modify theaerodynamics, but a uniform thickness provides more than adequateperformance.

For ease of assembly, especially for attachment to the inside of thehollow sleeve 14, the film 12 includes an extended tail 62 with atapered end 64 that allows easy insertion of the film 12 either into thesleeve 14, or in the case of a capillary application, into the plastictube or capillary 30. A pair of small wings 66 stick out when the film12 is inserted into the sleeve 14 and limit its travel into the sleeve14, providing reproducible positioning. For films to be attached to theoutside of the rod 16, a shorter, broader tail with a square end can beused. In both cases, the tail can be perforated with small apertures 68to improve gluing strength. In addition, in a controlled atmospherecapillary embodiment, the apertures 68 trap small liquid drops when thefilm 12 is placed in a crystal-containing drop that later serve tomaintain a humid environment in the capillary 30. For the thinnestpolyimide films, all right angle cuts in the design—which are points ofstress concentration—can be replaced with curves of finite radius toimprove film robustness against tearing.

To fabricate the patterned polyimide films, a number of differentprocesses can be employed, depending on. the desired crystal aperturesize and film thickness. A first, preferred film fabrication process isbased on photodefinable polyimide and can produce much smaller aperturesizes. To begin, a ½μm silicon dioxide layer, to be used as asacrificial layer during film lift-off, is deposited onto the surface ofa clean silicon wafer. The wafer is spin-coated with positive tonephotoexposable PWDC1000 polyimide. Standard processes such aspre-coating with an adhesion promoter or pre-baking in a vapor-primingoven ensure good adhesion between the wafer and polyimide. After a briefbake, the polyimide is soft-contact exposed through a chromium-glassmask containing the mount's pattern, and then submerged in developer toremove the exposed polyimide. The remaining patterned polyimide is thencured in a nitrogen-atmosphere oven. Finally, the wafer is submerged indilute HF to remove the sacrificial silicon dioxide layer, allowing thepatterned polyimide film to float free of the wafer. This process issuitable for fabricating polyimide films with thicknesses of 3-15 μm,which can then be patterned to an aperture size of 5 μm, which issuitable for mounting the smallest macromolecular crystals from whichdiffraction can currently be obtained. Thinner films and smalleraperture sizes can be obtained while maintaining adequate film rigidityby reducing the film's lateral dimensions and increasing its curvature.In a test setup using a 3 inch silicon wafer with a patterned polyimidefilm, 130 sample mounts can be produced at a time. With 8 inch wafersthis number can be increased to over 1000, making the individual mountsvery inexpensive to produce. Other common microfabrication processessuch as contact printing and spray (“inkjet”) printing could also beused, especially for mounts with larger aperture sizes.

For sample apertures larger than 50 μm, a fabrication process based onthe copper-polyimide-copper sheet material used for flexible electroniccircuits can be employed. A silicon wafer is spin-coated withphotoresist and a piece of this sheet material matching the wafer sizeis pressed on top using a piece of glass to ensure a flat surface. Thisassembly is then baked to cure the photoresist and firmly attach thecopper-polyimide-copper to the wafer. A second layer of photoresist isthen applied on top of the copper and the wafer baked a second time.This layer is exposed in a broad-band UV contact aligner through achromium glass mask containing the sample mount pattern (replicated tocover the wafer area) and then developed to remove the exposedphotoresist. The pattern is etched into the top copper layer usingferric chloride, and is then etched into the polyimide using an O₂—CHF₃gas plasma etch, after which the patterned copper-polyimide-copper filmdetaches from the wafer. The patterned sheet is placed in a bath ofShipley 1165 to remove residual photoresist and then into a secondferric chloride bath to etch away all remaining copper on the top andbottom of the polyimide film.

The minimum feature (e.g., aperture) size conveniently obtained by thesubject process is limited by the non-vertical etch profiles and by theminimum copper (9 μm) and polyimide (25 μm) thicknesses of commerciallyavailable flexible circuit materials to about 50 μm.

Optionally, the surface properties of the fabricated polyimide films canbe modified to improve the performance of the mounts in differentsolutions. Polyimide is naturally hydrophobic. In this state, the samplemounts tend to repel the mother liquor of soluble proteins and can causedisturbance of protein crystal-containing droplets. A broad variety oftechniques have been developed to modify the surface properties ofpolyimide. To make the subject polyimide films hydrophilic, thepreferred process is to expose them briefly to an oxygen gas plasma etchafter lift off and drying. This treatment minimizes drop disturbancesand allows crystals to be easily retrieved. However, with practiceuntreated mounts appear to perform nearly as well and reduce excessliquid, and their hydrophobic properties may be more useful for dropscontaining detergents.

Tests were conducted to compare the diffuse X-ray background versusresolution produced by a prior art 20 μm nylon loop mount and a 9 μmthick polyimide mount constructed in accordance with the preferredembodiments of the present invention. The tests established that themore rigid polyimide mount provides much less backgroundscatter—especially in the important 2-5 Å resolution range. In general,the scoop-like action together with the reproducibility of the sampleopening's size, shape and orientation make the subject mounts mucheasier to use than loops, especially for small crystals. As an example,the mounts have been used to mount and obtain a complete data set from a5-7 μm macromolecular crystal. The subject mounts can also be used forlarge crystals with sizes of hundreds of micrometers, provided thickerpolyimide films (10-25 μm) are used. Because of the small volume ofpolyimide in the beam path for all sample and mount orientations andpolyimide's low density and atomic number composition, its contributionto background scatter is small compared with that from disorderedinternal and external solvent even for very small crystals. This volumecan be reduced by optimizing the balance between film thickness (whichdetermines bending rigidity) and the width of polyimide around thesample aperture. The same basic design fabricated by a thin film processshould be applicable down to sample apertures of a few micrometers orless, provided the film thickness is reduced to account for the etchprofile and the lateral mount dimensions are reduced to preventbuckling. For extremely small crystals the aperture can be eliminated,and a small curved solid area at the tip can be used to retrieve andsupport the crystal.

Another major advantage of the mounts constructed in accordance with thepreferred embodiments is that they are much more rigid than loops. Theyremain rigid when submerged in crystal containing drops, simplifyingcrystal retrieval. They do not collapse during “annealing” or“tempering” procedures used to improve diffraction quality, and show noevidence of mosaicity broadening due to flutter in the cryostream.Finally, it should be noted that these microfabricated mounts should bemore generally useful for small crystals of all kinds, both organic andinorganic. For “dry” crystals the wicking aperture can be eliminated asnoted previously, and crystals can be firmly attached using glue orgrease, or using a small drop of an ethyl cellulose-ethyl acetatemixture which can be washed away after data collection.

Numerous additional variations or modifications could be made to thepreferred embodiments. For example, the films could be fabricated usingother polymers, or two or more layers of different polymers to givebetter rigidity. The surface of the film can be patterned to produce aribbed structure for better rigidity and bending characteristics withminimum total mass. In addition, the surface can be chemically treated,for example, to make the sample aperture water repellant and the“wicking” aperture water attractive. Pattern alignment marks can bedisposed on the film around the sample aperture to facilitate automatedalignment of the crystal. To facilitate different sized crystals, thesample aperture size can be made any size, and multiple sizes can bemade on a single polyimide sheet. The width of polyimide around theaperture can then be minimized for each crystal size, minimizing theamount of X-ray scattering from the aperture. Where the support includesuse of a cylindrical rod, the rod can be made hollow and of alow-specific heat, high thermal conductivity material to minimizethermal mass and evaporation of nitrogen during plunge cooling. Finally,the end of the rod where the film is attached can be tapered so that thefilm bends into part of a cone rather than part of a cylinder. Bycorrect choice of aperture size for a given sample size and by choosingthe angle of the rod's taper, the crystal can be placed on the axis ofrotation of the sample mount/goniometer.

In conclusion, the present invention comprises a new approach tomounting crystals for macromolecular cryocrystallography that maintainsthe many advantages of the nylon loops now in wide use and at the sametime resolves most of their deficiencies. Microfabricated polyimide filmsample mounts are better suited to handling the very small crystals thatcan now be characterized at state-of-the-art synchrotron beam lines andthat are being produced in abundance via automated crystallization atstructural genomics centers. These mounts should simplify automation ofX-ray data collection, and are better suited than existing alternativesfor automated crystal retrieval from liquid droplets. Because theirfabrication is based on standard microelectronics industry processes,these mounts should be easy and inexpensive to produce in quantities ofmillions per year that will soon be required by worldwide structuralgenomics efforts. They should be more generally useful for smallorganic/inorganic crystals of all sorts, and especially those of smallorganic/biological molecules that benefit from cryocooling for X-raydata collection.

Although the invention has been disclosed in terms of a number ofpreferred embodiment and numerous variations thereon, it will beunderstood that numerous additional modifications and variations couldbe made thereto without departing from the scope of the invention asdefined in the following claims.

1. A mount for holding a crystal to be examined using X-ray crystallography, said mount comprising: a film having a tip end for reception of a crystal; and means for imparting curvature to said film to increase the structural rigidity of said film.
 2. The mount of claim 1, wherein said film is formed of polyimide and has a thickness of 50 microns or less.
 3. The mount of claim 1, wherein said means for imparting curvature to said film comprises a rod having a curved outer surface to which said film is attached.
 4. The mount of claim 3, wherein said rod has a beveled or conical top end to increase a viewing angle for a sample crystal.
 5. The mount of claim 1, wherein said means for imparting curvature to said film comprises a first rod having a curved outer surface and a second, hollow rod into which said first rod is inserted, said second, hollow rod having a curved inner surface, wherein said film is inserted between said curved outside surface of said first rod and said curved inner surface of said second, hollow rod.
 6. The mount of claim 5, wherein said first and second rods each has a beveled or conical top end to increase a viewing angle for a sample crystal.
 7. The mount of claim 1, further including: a base to which said film is attached; and a plastic, semi-rigid, X-ray transparent tube that is pulled over said film and includes a first end affixed to said base in an air tight sealed manner; and, a second sealed end to facilitate controlling the environment to which a sample crystal is exposed.
 8. The mount of claim 1, wherein a first, sample aperture is disposed at said tip end of said film.
 9. The mount of claim 8, wherein said film further includes a second aperture spaced from said first, sample aperture and connected to said first aperture by a channel for wicking excess liquid from said first, sample aperture.
 10. The mount of claim 8, wherein said first, sample aperture is surrounded by a rim at said tip end of said film, said rim having a width of 100 microns or less.
 11. The mount of claim 1, wherein said film includes a second, tail end opposite to said tip end, said tail end being tapered to facilitate insertion of said film into a hollow mounting rod.
 12. The mount of claim 1, wherein said film further includes means for facilitating automated alignment of a sample crystal in an X-ray beam.
 13. The mount of claim 1, wherein said film is comprised of a plurality of polymer layers.
 14. The mount of claim 1, wherein said film is treated to produce a hydrophobic or hydrophilic surface.
 15. A mount for holding a crystal to be examined using X-ray crystallography, said mount comprising: a base having a cylindrical portion extending upwardly from said base; a sample crystal holder attached to said cylindrical portion; and a plastic, semi-rigid, X-ray transparent tube having: a first end pulled over said crystal holder and said cylindrical portion of said base, thereby making an air tight seal with said base; and, a second sealed end to facilitate controlling the environment to which a sample crystal is exposed.
 16. The mount of claim 15, wherein said crystal holder includes a film having a tip end for reception of a sample crystal and means for imparting curvature to said film to increase the structural rigidity of said film.
 17. The mount of claim 16, wherein said means for imparting curvature to said film comprises a hollow rod into which said film is inserted, said hollow rod being affixed to said cylindrical portion of said base.
 18. The mount of claim 16, wherein said film and said hollow rod are sized such that said hollow rod imparts curvature to said film when said film is positioned therein.
 19. The mount of claim 16, wherein said hollow rod includes an inner curved surface and means are provided for holding said film engagement with said inner curved surface.
 20. The mount of claim 16, wherein said hollow rod has a beveled top end to increase a viewing angle for said crystal. 